Type II diabetes is characterized by abnormal glucose homeostasis due to increased insulin resistance. Glucose metabolism leads to the formation of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), an indicator of metabolic status, and O-GlcNAc transferase (OGT) transfers O-GlcNAc to target proteins. Because O-GlcNAc affects protein function, Yang et al. investigated whether O-GlcNAc linked metabolism with insulin signaling. Phosphoinositide 3-kinase (PI3K)-mediated phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] generation in response to insulin recruits insulin receptor substrate (IRS) proteins and Akt to the plasma membrane to mediate IR signaling. The authors found that OGT, which does not contain any known PIP-binding domain, bound to PI(3,4,5)P3-coated beads, whereas the OGT mutant KK/AA, in which 2 C-terminal lysine residues were mutated to alanines, did not. Fluorescence imaging of COS-7 cells that expressed either OGT or KK/AA fused to green fluorescent protein (GFP) showed translocation of OGT-GFP, but not KK/AA-GFP, to the plasma membrane after treatment of starved cells with serum, which was blocked by inhibition of PI3K. Endogenous OGT colocalized with Akt at the plasma membrane of serum-treated cells. Adenoviral overexpression of OGT in adipocytes inhibited insulin-stimulated phosphorylation of Akt and increased inhibitory phosphorylation of IRS-1 on serine residues compared with control cells, modifications that are associated with decreased insulin responsiveness. Immunoprecipitation assays confirmed the modification of insulin signaling proteins by OGT. Mice whose livers were treated with adenovirus that expressed OGT had a higher abundance of plasma insulin, decreased basal glucose turnover, and decreased expression of insulin-dependent genes compared to those treated with adenovirus that expressed KK/AA. This study shows that OGT inhibits insulin responsiveness in a PI3K-dependent mechanism, leading the authors to suggest that this may play a role in the development of insulin resistance in type II diabetes.